Purpose:
High intraocular pressure (IOP) is often a risk factor for glaucoma. The ciliary body (CB) is an apically adherent epithelial structure responsible for aqueous humor secretion and thus IOP modulation. However, the regulation of its development and secretion remains poorly understood. This study reveals an important role of N-cadherin in the regulation of CB morphogenesis and a surprising role in the regulation of CB secretion.

Methods:
Trp1-cre was used to conditionally delete N-cadherin (Ncad) in both CB epithelial layers. Mutant phenotypes were characterized by standard procedures, including immunohistochemistry, mRNA in-situ hybridization, BrdU labeling, TUNEL labeling and Western blotting. Additionally, co-immunoprecipitation was performed to verify protein interactions. To further support our hypothesis, injection of Par3 shRNA lentiviruses into developing CBs was utilized to demonstrate the role of the cell polarity in CB morphogenesis.

Results:
Ncad mutant CBs show defective morphogenesis. Those mutant CBs maintain normal expression of the CB markers, Msx1, Otx1, Pax6 and Notch2, suggesting that N-cadherin is dispensable for CB specification. Our BrdU labeling results show that cell proliferation is drastically reduced in the Ncad mutant CBs. In addition, the cellular localization of the Par3/Par6/aPKC polarity complex is compromised in the Ncad mutant CBs, and shRNA-mediated knockdown of Par3 in the developing CB also disrupts its morphogenesis, suggesting that one of the N-cadherin functions in the regulation of CB morphogenesis is to maintain the epithelial polarity. Finally, secretion of Collagen IX is also reduced in the Ncad mutant CBs. Our co-IP results show that N-cadherin physically interacts with gap junction protein Connexin43, which is essential for aqueous humor secretion, indicating that N-cadherin regulates CB secretion by modulating the function of Connexin43.

Conclusions:
Our findings in this study have revealed two important roles of N-cadherin in the CB: regulation of CB morphogenesis and secretion. N-cadherin regulates CB morphogenesis at least in part by stabilizing the epithelial polarity and maintaining cell proliferation. N-cadherin also regulates aqueous humor secretion by directly modulating the function of the gap junction protein Connexin43. Therefore, this study has provided important insights into how CB morphogenesis and secretion are regulated at the molecular level.